Nucleic acid hybridization methods are currently used to detect the presence of nucleic acid regions known or suspected to be associated with the natural functioning of a living organism or nucleic acid residues obtained from various sources. Nucleic acid hybridization can also be used to detect sections of nucleic acid regions known or believed to be associated with an organism's disease state, metabolic state or life stage that the organism is experiencing during its life cycle. The accuracy of hybridization typically can be revealed during melting curve analysis of hybridized nucleic acid regions. Still further, there is a need for systems, methods, and compositions of matter to improve the specificity of binding between nucleic acid regions of nucleic acid sources.
Microarray technology has been the dominant genomics methodology but suffered from problems with repeatability and inaccuracy. In the quest for superior methods for genomics analysis, an abundance of next generation sequencing (NGS) methods were developed since the late 2000s. This allowed sequencing the human genome to drop in price from about $3 billion (2004) to approximately $20 k per genome as of early 2010. Genomics is certainly one of the fastest developing areas of the life sciences but large gaps continue to exist in the price performance of NGS in relation to other genomics techniques. While there have been dramatic pricing drops for the actual sequencing process, the price of NGS when used for gene expression profiling and SNP analysis is not competitive with microarrays that range in the hundreds of dollars per assay.
Furthermore, NGS techniques were developed for whole genome sequencing and sequence all DNA present in the sample. Analysis of specific parts of the genome or a subset of genes requires capture-enrichment assays. These consist of standard microarray chips which hybridize specific sequences but allow other unwanted sequences to be washed away. While, enrichment can be over 100 fold using this methodology, only between 30% to 60% of the captured DNA can come from the desired sections of the genome. As a result, capture enrichment assays typically are not very efficient and the depth or redundancy of sequence coverage varies with each experiment.
The melting curve microarray originated as a method for improving the accuracy of microarray gene expression profiling. The use of its technology is envisioned to simplify and lower cost for single nucleotide polymorphism (SNP) analysis. Melting curve analysis of double stranded DNA (dsDNA) has been practiced since the early 1960s in single tube reactions also referred to as liquid phase reactions. These experiments were done in tubes or liquid phase with the DNA free in solution. Since the discovery of melting analysis, the bulk of research has been spent studying liquid phase reactions. A common limitation to liquid phase melting curves is the inability to achieve one base pair resolution of detection. However, the application of melting curve analysis to the microarray or solid phase reaction is a relatively new and not completely understood process.
At the present time, there exists a need for a method and apparatus that can utilize measurement of the melting of target DNA away from probes bound to a glass microarray and that should distinguish between perfect match and mismatches on an individual probe spot and approximate the relative amounts of each species at a very low cost. Still further, it would be advantageous to have systems, methods and compositions for enhancing the specificity of nucleic acid hybridization. Further, it would be advantageous to have a method that can simultaneously analyze DNA sequence data while functioning as capture-enrichment tool, has sensitivity, is not time-consuming and is efficient, safe, and effective. Moreover, these methods, systems and compositions can be useful for improving the specificity of nucleic acid hybridization and their applications in health care, environmental research, pharmaceutical industry and food industry and are applicable for many other diagnostic, biotechnical and scientific purposes.